Comprehensive Validation of Cytology Specimens for Next-Generation Sequencing and Clinical Practice Experience

Abstract

Biopsy specimens are subjected to an expanding portfolio of assays that regularly include mutation profiling via next-generation sequencing (NGS). Specimens derived via fine-needle aspiration, a common biopsy technique, are subjected to a variety of cytopreparatory methods compared with surgical biopsies that are almost uniformly processed as formalin-fixed, paraffin-embedded tissue. Therefore, the fine-needle aspiration-derived specimens most commonly accepted for molecular analysis are cell blocks (CBs), because they are processed most similarly to surgical biopsy tissue. However, CB preparations are fraught with challenges that risk unsuccessful sequencing and repeat biopsies, with the potential to further increase health care costs and delay clinical care. The diversity of cytopreparations and the resource-intensive clinical validation of NGS pose significant challenges to more consistent use of non-CB (NCB) cytology specimens. As part of clinical validation of a targeted NGS assay, DNA subjected to nine cytopreparatory methods was evaluated for sequencing performance and was shown to be uniformly acceptable for clinical NGS. Of the 379 clinical cases analyzed after validation, the majority (56%) were derived from NCB cytology specimens. This specimen class had the lowest DNA insufficiency rate (1.5%) and showed equivalent sequencing performance to surgical and CB formalin-fixed, paraffin-embedded tissue. NCB cytology specimens are valuable sources of tumor nucleic acid and are the preferred specimen type for clinical NGS at our institution.

Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis

“…The development of liquid-based cytology (LBC) supports the availability of cytology samples for both cytopathology and genome biology. Furthermore, comprehensive genome analysis of LBC samples has been attempted recently in the fields of pathology and laboratory medicine [13, 14]. Studies have reported that DNA extracted from conventional cytological or LBC samples could be applied to NGS analysis [15–18], but these studies did not always include detailed descriptions of their fixation and DNA extraction methods.…”

Robust assessment of tumor mutational burden in cytological specimens from lung cancer patients

“…Cytological specimens represent an important fraction of lung cancer samples and occasionally the only source of tumor cells, as in pleural effusions [25]. Moreover, cytological smears are fixed in alcohol-based solutions, and their DNA is considered to have comparable quality to DNA obtained from fresh-frozen tissue [26]. Because little is known about TMB values derived from cytological samples, we evaluated the feasibility of cytological smears as the DNA source for TMB estimation and compared it to FFPE samples.…”

Expediting Comprehensive Molecular Analysis to Optimize Initial Treatment of Lung Cancer Patients With Minimal Smoking History

“…In support of the promise of this strategy, two groups recently showed that NGS can be performed on the fine needle aspirate or cell-free DNA isolated from the supernatant of the aspirate. 14,15 Molecular testing can also be hastened by analyzing tumor-derived genetic material from non-tissue-based sources. For example, plasma analysisa process that can be performed in 7 to 10 daysis increasingly used to genotype tumors at diagnosis where it has been shown to be highly concordant with tissue-based testing.…”